Molecular Docking and ADME-TOX Profiling of Moringa oleifera Constituents against SARS-CoV-2

Highlights What are the main findings? Bioactive compounds of Moringa oleifera exhibited activity against SARS-CoV-2. Computational approaches to studying the antiviral activity of natural compounds against SARS-CoV-2 might be a time- and money-saving option in the drug discovery and development process. What is the implication of the main finding? The antiviral potential of Moringa oleifera against SARS-CoV-2 may contribute to an advanced level of pharmaceutical research. Advanced computational methods can be used to search for novel anti-SARS-CoV-2 agents from natural products. Abstract The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2019) etiological agent, which has a high contagiousness and is to blame for the outbreak of acute viral pneumonia, is the cause of the respiratory disease COVID-19. The use of natural products grew as an alternative treatment for various diseases due to the abundance of organic molecules with pharmacological properties. Many pharmaceutical studies have focused on investigating compounds with therapeutic potential. Therefore, this study aimed to identify potential antiviral compounds from a popular medicinal plant called Moringa oleifera Lam. against the spike, Mpro, ACE2, and RBD targets of SARS-CoV-2. For this, we use molecular docking to identify the molecules with the greatest affinity for the targets through the orientation of the ligand with the receptor in complex. For the best results, ADME-TOX predictions were performed to evaluate the pharmacokinetic properties of the compounds using the online tool pkCSM. The results demonstrate that among the 61 molecules of M. oleifera, 22 molecules showed promising inhibition results, where the compound ellagic acid showed significant molecular affinity (−9.3 kcal.mol−1) in interaction with the spike protein. These results highlight the relevance of investigating natural compounds from M. oleifera as potential antivirals against SARS-CoV-2; however, additional studies are needed to confirm the antiviral activity of the compounds.


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Computational approaches to studying the antiviral activity of natural compounds against SARS-CoV-2 might be a time-and money-saving option in the drug discovery and development process.
What is the implication of the main finding? • The antiviral potential of Moringa oleifera against SARS-CoV-2 may contribute to an advanced level of pharmaceutical research.

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Advanced computational methods can be used to search for novel anti-SARS-CoV-2 agents from natural products.

Abstract:
The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2019) etiological agent, which has a high contagiousness and is to blame for the outbreak of acute viral pneumonia, is the cause of the respiratory disease COVID-19.The use of natural products grew as an alternative treatment for various diseases due to the abundance of organic molecules with pharmacological properties.Many pharmaceutical studies have focused on investigating compounds with therapeutic potential.Therefore, this study aimed to identify potential antiviral compounds from a popular medicinal plant called Moringa oleifera Lam.against the spike, M pro , ACE2, and RBD targets of SARS-CoV-2.For this, we use molecular docking to identify the molecules with the greatest affinity for the targets through the orientation of the ligand with the receptor in complex.For the best results, ADME-TOX predictions were performed to evaluate the pharmacokinetic properties of the compounds using the online tool pkCSM.The results demonstrate that among the 61 molecules of M. oleifera, 22 molecules showed promising inhibition results, where the compound ellagic acid showed significant molecular affinity (−9.3 kcal.mol−1 ) in interaction with the spike protein.These results highlight the relevance of investigating natural compounds from M. oleifera as potential antivirals against SARS-CoV-2; however, additional studies are needed to confirm the antiviral activity of the compounds.

Introduction
In December 2019, a new respiratory disease called Noble Coronavirus Disease 2019 (COVID-19) was identified in Wuhan, China.The etiologic agent involved is the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1].It is a positive-sense RNA beta-coronavirus [2] that doesn't have any segments and has a high rate of spreading.It is what caused the acute viral pneumonia outbreak.
In recent years, the use of medicinal plants in the treatment of various diseases has increased because this approach has gained credibility as a result of important pharmaceutical research [3,4].Among these plants, we highlight the genus Moringa, the only representative of the Moringaceae family, which comprises fourteen species widely distributed in the tropical regions of the planet [5,6].Among the species described for the genus is Moringa oleifera Lam.In Brazil, it was introduced as an ornamental plant around 1950 [7], and since then, it has been widely cultivated due to its high nutritional value, especially the leaves, which are rich in carotene, ascorbic acid, and iron [8,9].
All parts of M. oleifera are traditionally used for different purposes, but the leaves are the most commonly used [10].These can be consumed directly, raw and dried, or in the form of an aqueous infusion to treat various ailments, including malaria, typhoid fever, parasites, arthritis, swelling, skin diseases, hypertension, and diabetes.In addition to being used to induce lactation and improve the immune system, M. oleifera is characterized by having a high concentration of proteins, vitamins, minerals, β-carotene, and secondary metabolites with antioxidant properties, including glucosinolates, flavonoids, and phenolic acids, which have effects against chronic diseases [11,12].This plant is easily found in tropical and subtropical regions; therefore, these classes of people are very popular with it [13].
The computer techniques used in bioinformatics help procedures in this field because they save time and money and speed up the process of obtaining results from experiments in vitro and in vivo.This is because they make it easier to organize data and help choose targets or hypotheses to be tested on the bench [14][15][16].These approaches are essential for identifying promising compounds with pharmacological potential for the development of new drugs.In this way, finding a promising treatment becomes a top priority, and it is important to use computational methods to quickly find compounds that have a molecular affinity for the proteases of the SARS-CoV-2.
Several studies were done to scientifically prove that Moringa can be used to treat these illnesses because it is thought to have healing properties [17][18][19].Different parts of the tree, like the root, bark, gum, leaves, fruits (pods), and flowers, have different health benefits.For example, they can help with allergies, fight cancer, lower blood sugar, fight fungal growth, protect the liver, and boost the immune system [20].
Muratov et al., (2021) [21] said that we need to carefully look at computational approaches in order to find effective treatments for SARS-CoV-2.The pandemic motivated global efforts to identify therapeutic approaches, with an emphasis on computational research.Effective integration of these tools with experimentation is crucial for validating results and developing antiviral therapies.This underscores the need for a multidisciplinary approach where computational research identifies drug candidates and clinical trials validate these findings.
Compounds derived from natural sources have significant therapeutic value and make up more than half of the drugs approved by the FDA [22].Natural products represent a valuable source of bioactive molecules for drug screening.To date several studies report that natural products have anti-SARS-CoV-2 effects.The virtual screening of natural products using the molecular docking method plays a crucial role in evaluating the inhibitory activity of these molecules against SARS-CoV-2.However, these findings should be validated through in vitro studies [23].Power et al., (2022) [24] screened ten natural compounds through in silico studies and found favorable ADMET profiles for the in vitro evaluation of their activity against SARS-CoV-2.During the in vitro analysis, four compounds were active against SARS-CoV-2, suggesting the application of in silico studies in in vitro evaluation in the drug discovery field.
The compounds present in the plant are promising for research aimed at finding substances that will have good results against the essential proteins of SARS-CoV-2.With the help of tools in the molecular docking method, it is possible to figure out the best way for the compound and the target protein to connect at the molecular level [25].This function lets you figure out how the compound acts in the active site of a pathogen's key protein and see the molecular interactions between the compound and the protein [26].This tool also allows virtual drug screening and the characterization of molecular structures [27].Thus, this study aims to identify compounds with inhibitory potential based on the mechanisms of action in complex with SARS-CoV-2 targets and to make predictions of the absorption, distribution, metabolization, excretion, and toxicity (ADME-TOX) of M. oleifera compounds.

Selection of Binders
A total of 61 chemical compounds from the M. oleifera species were selected, covering classes such as flavonoids, alkaloids, terpenoids, coumarins, and saponins, among others, in national and international scientific databases such as Scientific Electronic Library Online (Scielo), National Center for Biotechnology Information (PubMed), Elsevier Group (Scopus and Science Direct), and Google Scholar.The search was conducted using the keyword "Moringa oleifera" in combination with "chemical constituents" or "phytochemicals".The corresponding chemical structures were acquired through the Pubchem platform (http: //pubchem.ncbi.nlm.nih.gov/,accessed on 3 September 2022).

Molecular Docking
The 3D structures of three coronavirus targets were obtained from the Protein Data Bank (PDB) database (http://www.rcsb.org/,accessed on 10 October 2022) [28], with the respective codes: spike protein (PDB ID: 6VXX), angiotensin converting enzyme-ACE2 (PDB ID: 1R42), main protein Mpro (PDB ID: 6LU7), while the Receptor5 (RBD-spike/ACE2 interaction site) was designed by Barros et al., (2020) [29].For molecular affinity, they were prepared by removing all water molecules and other groups, such as ions, using Chimera V software.13.1 [30].Afterwards, polar hydrogen atoms were added, the Gasteiger partial charges were calculated, and the non-polar hydrogens were merged in both parts (protein and ligand) using the Autodock Tools (ADT) program version 1.5.6.Subsequently, the docking was carried out using the program AutoDock Vina [31].The grid box size was set to 30 Å for each axis.The grid boxes were centered on the coordinates of the atoms of the residues located in the active site region and interface region, namely: Gly548(A) (6VXX), His374 (A) (1R42), Gly143(A) (6LU7), and Phe32(B) (Receptor 5) (Table 1).The number of modes was set to 50, and the exhaustiveness was set to 24.With the LIGPLOT program, 2-D diagrams of protein-ligand complexes were made from the PDB file, which was the standard input.Pictures were made to show where the hydrogen and hydrophobic bonds of the compounds interact with the amino acids of the viral proteins [32].The analyses were concentrated on the lower energy complexes of the socket conformation.The lowest energy conformations, combined with visual inspection, were chosen for a more detailed analysis [16].
In this study, the PDB structures of three proteins were selected as main targets for virtual screening of compounds aimed at discovering potential antiviral agents against SARS-CoV-2: the main protease (Mpro) (PDB ID: 6LU7), the spike glycoprotein (S) (PDB ID: 6VXX), and the functional receptor ACE2 (PDB ID: 1R42).This selection was based on their recognized importance in the virus life cycle and infection mechanism.M pro was selected as a target due to its essential function in viral replication.The main protease (M pro ) is responsible for the cleavage of the viral polyprotein into independent functional proteins, which is necessary for the replication of the SARS-CoV-2 virus [33].Its inhibition can effectively halt the viral replication process, making it a promising target for antiviral therapies.Previous studies highlighted the relevance of Mpro as a therapeutic target, and the PDB structure of M pro (PDB ID: 6LU7) was used as the basis for our molecular docking simulations [34,35].
The spike (S) glycoprotein was also chosen as a target due to its fundamental role in virus entry into host cells.The spike protein's receptor-binding domain (RBD) connects with the host cell's functional ACE2 receptor, which helps the virus infect the cell [36].Existing research indicates that the spike is a relevant target for the search for inhibitors, as interrupting this early stage of the viral cycle is an effective strategy for controlling infection.The PDB structure of spike (PDB ID: 6VXX) was used as a reference for our molecular docking analyses [37,38].
The functional ACE2 receptor was also considered an important target, as it plays a central role in the binding and internalization of SARS-CoV-2 into host cells [39].Inhibition of ACE2 can block the interaction between the viral spike and the host cell, preventing infection.Studies on inhibitors against SARS-CoV-2 included ACE2 as a molecular target, aiming to interrupt the COVID-19 infection process [37,40].Therefore, these proteins were chosen as research targets because they play a key role in the life cycle of viruses and could be used as targets for antiviral drugs.

Molecular Docking
M. oleifera plants were tested through the molecular docking process with four receptors that are essential in the process of viral infection and replication.Among the receptors, two proteins are from the virus: the spike glycoprotein and the main protein (M pro ).In addition to these, ACE2 (angiotensin converting enzyme) and receptor 5 (RBD) were also used.
A total of 244 dockings were performed with the 61 compounds and the four receptors.The results show an energy variation from −3.4 to −9.3 kcal/mol.The lower the binding energy between the compound and the receptor, the better the complex interaction (Table 2).To select the best energy parameters, interactions smaller than −7.9 kcal/mol were considered, thus obtaining the 22 interactions described in Table 3.The lowest binding energy (−9.3 kcal/mol) was obtained through the interaction of the ellagic acid compound with the spike protein of SARS-CoV-2 (Figure 1).The complex formed six hydrogen bonds with amino acids: Asn978, Leu977, Arg1000, Tyr741, Met740, and Thr549, and four hydrophobic interactions: Phe541, Val976, Gly744, and Gly548.Figure 2 shows all docking performed in this study, with binding energies ranging from −3.4 to −9.3 kcal/mol.The group that obtained the most interactions was group B, which presented energies from −4.0 to −4.9 kcal/mol.Two groups, F and G, had the lowest number of molecular interactions, 22 in total, with energies ranging from −8.0 to −8.9 kcal/mol and from −9.0 to −9.3 kcal/mol, respectively.However, these were the results of the molecular interaction of the ligands with the targets, which was considered more satisfactory in this study (Table 3).Figure 2 shows all docking performed in this study, with binding energies ranging from −3.4 to −9.3 kcal/mol.The group that obtained the most interactions was group B, which presented energies from −4.0 to −4.9 kcal/mol.Two groups, F and G, had the lowest number of molecular interactions, 22 in total, with energies ranging from −8.0 to −8.9 kcal/mol and from −9.0 to −9.3 kcal/mol, respectively.However, these were the results of the molecular interaction of the ligands with the targets, which was considered more satisfactory in this study (Table 3).The best molecular interactions with the receptors shown in Figure 3 come from 14 ligands (Figure 4).Some of these ligands had good interactions with more than one protein.It was the spike protein that stood out as the receptor with the most effective complex interactions.It made connections with 16 different compounds.This protein is important for a virus to get into a cell because it interacts with ACE2.Two ligands worked well for the anchoring process with Mpro, which is in charge of virus replication.However, only one compound interacted with ACE2, which is the cell receptor that lets the SARS-CoV-2 virus into the body.Finally, three coupling results with RBD were obtained.The best molecular interactions with the receptors shown in Figure 3 come from 14 ligands (Figure 4).Some of these ligands had good interactions with more than one protein.It was the spike protein that stood out as the receptor with the most effective complex interactions.It made connections with 16 different compounds.This protein is important for a virus to get into a cell because it interacts with ACE2.Two ligands worked well for the anchoring process with Mpro, which is in charge of virus replication.However, only one compound interacted with ACE2, which is the cell receptor that lets the SARS-CoV-2 virus into the body.Finally, three coupling results with RBD were obtained.The best molecular interactions with the receptors shown in Figure 3 come from 14 ligands (Figure 4).Some of these ligands had good interactions with more than one pro tein.It was the spike protein that stood out as the receptor with the most effective com plex interactions.It made connections with 16 different compounds.This protein is im portant for a virus to get into a cell because it interacts with ACE2.Two ligands worked well for the anchoring process with Mpro, which is in charge of virus replication.How ever, only one compound interacted with ACE2, which is the cell receptor that lets the SARS-CoV-2 virus into the body.Finally, three coupling results with RBD were obtained    Rutin, isoquercitrin, and lutein were the molecules that interacted best with proteins ACE2, M pro , and RBD during the molecular docking process.Figure 5 shows that the compound rutin formed the complex with the cellular protein ACE2 that had the lowest interaction energy.It had an interaction energy equal to binding of −8.2 kcal/mol.Seven amino acids (Glu398, Tyr385, Asp382, Asp350, Ala348, Ser47, and Ser44) and seven amino acids (Arg514, Asn394, Thr347, Trp349, Phe40, His401, and Glu402) had hydrogen bonds with it.
Several studies have been done on the pharmacological properties of different natural compounds and how they can be used to treat and prevent different diseases.These are important for the study and development of promising therapeutic candidates for various diseases.In this study, the molecular docking method was used.This method shows how the complex interacts with the macromolecule and how the compound blocks the action of the macromolecule.Through this method, the ligands ellagic acid, rutin, myricetin, luteolin, and quercetin, as well as other bioactive compounds that, after analyzing the in-silica results (Table 3), showed satisfactory binding energies in their interactions with the receptors, were shown to have inhibitory effects on the molecular targets spike, M pro , ACE2, and RBD, thus presenting antiviral effects against SARS-CoV-2.This is a preliminary study regarding activity against the virus.Further tests and in-depth studies about these compounds are needed in order to obtain more knowledge about their properties as promising potential therapeutic candidates in the treatment of this disease.Several studies have been done on the pharmacological properties of different natural compounds and how they can be used to treat and prevent different diseases.These are important for the study and development of promising therapeutic candidates for various diseases.In this study, the molecular docking method was used.This method shows how the complex interacts with the macromolecule and how the compound blocks the action of the macromolecule.Through this method, the ligands ellagic acid, rutin, myricetin, luteolin, and quercetin, as well as other bioactive compounds that, after analyzing the in-silica results (Table 3), showed satisfactory binding energies in their interactions with the receptors, were shown to have inhibitory effects on the molecular targets spike, M pro , ACE2, and RBD, thus presenting antiviral effects against SARS-CoV-2.This is a preliminary study regarding activity against the virus.Further tests and in-depth studies about these compounds are needed in order to obtain more knowledge about their properties as promising potential therapeutic candidates in the treatment of this disease.In Brazil, the National Health Surveillance Agency (ANVISA), the government agency responsible for controlling, monitoring, inspecting, and regulating the production, distribution, and marketing of medicines in the country, granted approval for the emergency use of six medicines intended for the treatment of COVID-19 as of June 2021.Among these drugs, remdesivir, paxlovid (nirmatrelvir + ritonavir), molnupiravir, and In Brazil, the National Health Surveillance Agency (ANVISA), the government agency responsible for controlling, monitoring, inspecting, and regulating the production, distribution, and marketing of medicines in the country, granted approval for the emergency use of six medicines intended for the treatment of COVID-19 as of June 2021.Among these drugs, remdesivir, paxlovid (nirmatrelvir + ritonavir), molnupiravir, and baricitinib stand out, being also recommended by the International Solidarity Initiative, led by the World Health Organization (WHO) [50,51].The results obtained from the interaction of these drugs with SARS-CoV-2 receptors are described in Table 4. Showing that none of the drugs showed results lower than −8.0 kcal/mol.We can observe that the drugs remdesivir (−7.9 kcal/mol) and paxlovid (−7.6 kcal/mol) had a greater interaction with M pro .The bioactive compounds from the Moringa species were looked at in this study.Isoquercitrin and rutin interacted better with M pro than the two drugs, with −8.9 and −8.8 kcal/mol, respectively.In addition to these, kaempferol (−7.8 kcal/mol), glucomoringin (−7.9 kcal/mol), and apigenin (−7.7 kcal/mol) also showed better results than paxlovid.

ADME-TOX Prediction
A computer method called pharmacokinetic prediction in silico is used to look into the ADMET properties of naturally occurring organic molecules that make living things work.This evaluates the absorption, distribution, metabolism, excretion, and toxicity of the molecules.The absorption prediction parameters of the compounds that obtained satisfactory binding energies with the SARS-CoV-2 targets are described in Table 5.
By looking at the molecules' characteristic absorption parameter, it was shown that most of the compounds tested could dissolve in water within the range of −1 to −5 (mol/L).This means that the compounds have a good hydrophilic capacity.Except lutein, brassicasterol, stigmasterol, and ergosterol, which presented values below −6 (mol/L).Regarding skin permeability, compounds with values above −2.5 cm/h are considered to have low skin permeability.All compounds showed values in the range of −2.799 cm/h to −2.735 cm/h, which indicates that all molecules are considered permeable to the skin.Apigenin had a permeability value of 1076 cm/s, brassicasterol had a value of 1209 cm/s, stigmasterol and ergosterol each had a value of 1.21 cm/s, and lutein had a value of 1284 cm/s.Such compounds had values greater than 0.90.The other compounds did not present satisfactory results in this regard.Of the analyzed compounds, only three inhibited P-gp I (brassicasterol, stigmasterol, and ergosterol), while four compounds inhibited P-gp II (lutein, brassicasterol, stigmasterol, and ergosterol).Importantly, the same compounds inhibited P-gp I and II.
Analysis of human intestinal absorption (AIH) is one of the most important ways to judge new drug candidates.The vast majority of molecules analyzed demonstrated an intestinal absorption range between 72.5 and 94.7%, which indicates effective absorption.Isorhamnetin, kaempferol, lutein, catechin, apigenin, epicatechin, brassicasterol, stigmasterol, and ergosterol are some of the molecules that are in this group.That being said, it was seen that compounds like glucomoringin, isoquercitrin, rutin, and myricetin did not absorb well in the intestines.
The VDss (steady-state volume of distribution) is the theoretical volume at which a drug dose needs to be uniformly distributed to result in the same concentration as in blood plasma.The VDss is considered low for values below 0.71 L/kg and high for values above 2.81 L/kg.All compounds showed low VDss; that is, they are all more likely to be distributed in plasma than in tissues.
Regarding the potential for penetration of the blood-brain barrier (BBB), most compounds have a low potential to cross it.The compounds that were able to cross it showed values with logBB > 0.3; these were brassicasterol, stigmasterol, and ergosterol.Data on how cytochrome P450 proteins (CYP) interact show that some molecules block CYP1A2, CYP2C19, and CYP2C9 from breaking down other drugs in the body.In the study at hand, it was found that apigenin blocks three enzymes (CYP1A2, CYP2C19, and CYP2C9), while ellagic acid, myricetin, quercetin, luteolin, and isorhamnetin only block CYP1A2.None of the evaluated molecules showed inhibition of the CYP2D6 and CYP3A4 proteins or the CYP2D6 substrate.On the other hand, the compounds lutein, brassicasterol, stigmaterol, and ergosterol inhibited the CYP3A4 substrate (Table 6).Lethal concentration values (LC 50 ) represent the concentration of a molecule required to cause 50% of the flathead minnows to die.According to this study, it can be demonstrated that isoquercitrine might be most harmless, while catechin and epicatechin might be harmful, since the higher the lethal dose, the lower the degree of toxicity.Chronic oral toxicity in rats (LOAEL) is analyzed in the same way.Defined as the lowest dosage for observation of adverse effects, it had its most significant result for the compound rutin, which can be ingested in substantial amounts without causing chronic diseases.
The recommended maximum tolerated dose (MRTD) provides an estimate of the threshold toxic dose of chemicals in humans.For the analysis, an MRTD lower than or equal to 0.477 log (mg/kg/day) is considered low.The compounds lutein, glucomoringin, brassicasterol, stigmasterol, and ergosterol showed low values; therefore, they have low toxicity, while the other analyzed compounds showed high values.
The analysis demonstrates whether a given compound is likely to be a hERG I/II inhibitor.So, the analysis showed that no compound can stop hERG I from working.However, seven compounds were found to stop hERG II from working.These compounds are rutin, isoquercitrin, lutein, apigenin, brassicasterol, stigmasterol, and ergosterol.

Discussion
With the advent of COVID-19, there was a need to identify new substances with antiviral properties against SARS-CoV-2.Faced with this urgency, several clinical studies explored the concept of repositioning existing drugs as an agile approach to developing a new, effective therapeutic model.This scenario provided an opportunity to investigate alternative treatments, with an emphasis on the potential use of medicinal plants [52].
The study's main goal is to find compounds from M. oleifera that might be good at stopping the activity of SARS-CoV-2 targets, like the spike protein, Mpro, ACE2, and RBD, which would then lower or stop the virus from replicating.It is notable that previous studies searched for effective inhibitors of natural origin from plants with pharmacological activity, which are known in the literature for their use in the treatment and cure of various diseases.
In this study, molecular docking was performed to investigate the antiviral activity of M. oleifera compounds against SARS-CoV-2.The compound with the most negative binding activity to target proteins is predicted to play an essential role.The findings show that rutin had strong molecular interactions with all of the targets that were tested, and isoquercitrin had interactions with three targets, which were spike, M pro , and RBD.Thus showing that these compounds can be promising antiviral inhibitors against more than one target of interest in the search for therapeutics against the virus.
The outcomes indicate that five chemicals (luteolin, myricetin, ellagic acid, and rutin) had the worst molecular interactions with the spike protein.The ellagic acid that had the lowest binding affinity index in this study has a number of medical benefits, such as protecting cells from damage, reducing inflammation, and protecting nerves and the liver [53].According to studies, the compound also has strong anticancer activity [54], as well as other important biological functions like chemoprevention and antiviral activities [55].It has also been shown to stop mutations and reduce inflammation in bacteria and mammals.
Rutin is a flavonoid phytochemical compound present in a variety of plants with pharmacological properties for the prevention of various diseases.Its bioactive effects include antiviral [56], anti-asthma [57], antimicrobial [58], anti-inflammatory [59], and antioxidant activities [60].On the other hand, myricetin is a compound widely found in various human foods and beverages and is known for its diverse pharmacological properties, including antioxidant, anti-inflammatory, and anticancer effects [61], being antitumor [62], antibacterial [63], and antiviral [64,65].
Quercetin is also a flavonoid found in several plants and is considered a potent natural compound with biological properties.In silico and in vitro studies show that this compound has many health benefits, including fighting cancer, reducing inflammation, lowering blood pressure, preventing diabetes, reducing allergies, lowering cholesterol, preventing blood clots, and boosting mood [66][67][68].Thanks to its pharmacological properties, the luteolin compound is found in many plants that people eat and that are used in traditional medicine to treat a wide range of illnesses.The compound has many biological effects [69], such as anti-inflammatory [70], antioxidant [71], anticancer [72], antibacterial [73], and antiviral [74] properties.
Many virtual screening studies of natural compounds were conducted to evaluate their antiviral activity against SARS-CoV-2.This research covers a wide range of natural compounds, including polyphenols and flavonoids, and reveals the antiviral potential of these substances.They demonstrated the ability to inhibit the main proteases of the virus, which positions them as promising therapeutic agents for the treatment of COVID-19 [75].According to Aini et al., (2022) [76], in their in silico study on bioactive compounds with potential against SARS-CoV-2, the compounds ellagic acid and myricetin were identified as candidates that meet Lipinski's criteria, suggesting their viability as anti-SARS-CoV-2 agents.Our results are similar regarding the antiviral potential of these bioactive compounds.However, it is essential to highlight that additional investigations are necessary to substantiate and validate these results.
In silico studies conducted by Mawaddani et al., (2022) [77] on M. oleifera also suggest that this herb might be a potential candidate against SARS-CoV-2 infection.In this study, quercetin was believed to act against SARS-CoV-2, possibly through inhibiting viral entry and binding to the active sites of both the main protease (M pro ) and RNA-dependent RNA polymerase (RdRp) of it, demonstrating quercetin as a potential drug candidate against SARS-CoV-2.Inhibition of targets in the SARS-CoV-2 life cycle plays a crucial role in blocking essential processes required for virus life cycle progression, resulting in infection control.The main access route of SARS-CoV-2 to cells occurs through the interaction of the spike (S) protein with the ACE2 receptor [78], which is an essential component of the SARS-CoV-2 nucleocapsid and plays a fundamental role in recognizing cellular receptors.Thus, the interaction between protein S and ACE2 facilitates the cell membrane fusion process, allowing the virus to enter cells [79,80].Compounds with the ability to inhibit the interaction between the S protein and ACE2 can prevent the fusion process, resulting in blocking virus entry.
It's important to note that only a few of the natural products that were tested against the receptor binding domain (RBD) of SARS-CoV-2 were able to stop the spike protein from interacting with its receptor ACE2.Some of these molecules, like nimbin, curcumin, withaferin A, mangiferin, piperine, thebaine, andrographolide, and berberine, were found to be good at stopping this process [81].
The term "major protease", or M pro , is used due to its critical function in coronavirus gene expression and replicase processing [33].Based on the results obtained, the compounds b-amyrin and stigmasta-5,22-dien-3-ol demonstrated potential as main protease inhibitors (M pro ) of SARS-CoV-2.From the ADMET predictions and the assessment of biological activity, it is possible to safely infer that these compounds have the ability to exhibit antiviral activity [34].Several studies are focused on the search for SARS-CoV-2 M pro inhibitors, as inhibition of this enzyme has the potential to block viral replication, making it an attractive target for the development of antiviral drugs against SARS-CoV-2.
The drug repositioning strategy, which is a standard method used in pharmaceutical development research, tries to find new medical uses for drugs that have already been approved or are still in the testing phase [82].Several studies were conducted with the purpose of exploring the application of medicines already available on the pharmaceutical market as an alternative approach to combating SARS-CoV-2 [83].The drugs that stand out are ombitasvir and ledispavir [84], as well as chloroquine, atazanavir, and oseltamivir [85].Other drugs that stand out are baricitinib, molnupiravir, remdesivir, and paxlovid [86].
The drugs baricitinib, molnupiravir, remdesivir, and paxlovid were tested in clinical trials to see how well they could fight COVID-19 [87].Based on the results, Anvisa gave these drugs the green light to be used to fight the disease [50].When the molecular affinity between these drugs and the SARS-CoV-2 targets was looked at, it was seen that natural compounds from the M. oleifera plant had lower binding energy values than these drugs.This indicates a remarkable inhibitory activity of the natural compounds towards the tested targets.
Oral administration and high solubility are important parts of drug discovery plans for full absorption.Low solubility, on the other hand, limits absorption in the digestive tract.Predictions of pharmacokinetic and toxicity parameters (ADME-TOX) revealed that the majority of compounds demonstrated reasonable water solubility.All molecules proved to be permeable to the skin.The assessment of skin permeability is essential to understanding the ability of a molecule to cross the layers of the epidermis and dermis, which is relevant in the development of transdermal drug delivery systems [88].
As a way to test how well water-soluble drugs dissolve and pass through cells, Caco-2 cells were created.These cells are grown in transwell cell culture plates and come from a type of human colon cancer.This made it possible to predict how quickly they would absorb after oral administration [89].Notably, the compounds apigenin and ergosterol had a lot of permeability in Caco-2 cells, and they also had a lot of permeability in the mouth.
One of the main parameters for evaluating new drug candidates is the analysis of human intestinal absorption (AIH), in which molecules with absorption values between 70% and 100% indicate good intestinal absorption [90,91].The intestine generally represents the main site of absorption for orally administered medications, and most of the molecules analyzed showed potential for intestinal absorption.Among these molecules, ellagic acid, quercetin, luteolin, isorhamnetin, kaempferol, lutein, catechin, apigenin, epicatechin, brassicasterol, stigmasterol, and ergosterol stand out.
The steady-state volume of distribution (VDss) is a theoretical parameter that estimates how much of a drug needs to be spread out evenly in order to reach the same concentration as blood plasma [34].It was observed that all analyzed compounds have a greater probability of distribution in plasma compared to tissues.In the context of distribution parameters, the BBB plays a fundamental role in protecting the brain against harmful substances.The ability of a drug to cross this barrier is a critical criterion to be considered to reduce side effects, toxicities, or improve the effectiveness of pharmacological treatments in the brain [92].Our results suggest that only the compounds brassicasterol, stigmasterol, and ergosterol have the ability to cross the BBB.However, it is important to note that these compounds also demonstrated inadequate water solubility and inhibited both P-glycoprotein I and P-glycoprotein II.
The P-glycoprotein (P-gp), responsible for the absorption, distribution, metabolism, and excretion of several drugs [93], is an ATPase transmembrane that plays a significant role as a defense mechanism against harmful agents, promoting the pumping of toxins and xenobiotic substances out of cells.This P-gp plays a vital role as a biological barrier by expelling toxins and xenobiotics out of cells, thus protecting cell integrity [88,93].These results indicate that although these compounds have the ability to access the brain, they may present additional challenges in terms of bioavailability and interactions with transporter proteins.
A protein called OCT2 (organic cation transporter 2) is very important for the absorption, distribution, and renal clearance of many different drugs.Assessment of a drug candidate's ability to be transported by OCT2 provides valuable information not only about its elimination but also about possible contraindications [92].Our in silico analyses revealed that none of the evaluated compounds are substrates of human OCT2.This result is relevant in the context of the excretion of cationic molecules and suggests that these compounds may not interact significantly with the transport system mediated by OCT2 in the human body.
The Ames toxicity test is a method used to evaluate the mutagenic potential of a compound using bacteria [41,94].The results obtained are negative for most compounds.These results indicate that these compounds did not demonstrate toxicity in the test and, therefore, do not have mutagenic or carcinogenic potential.This is an important finding, as it suggests that these compounds can be considered safe in terms of mutagenic toxicity.
In this study, we used in silico approaches to show that compounds from M. oleifera might be useful in COVID-19.We used molecular docking assessments and ADME-TOX predictions to evaluate their therapeutic potential against COVID-19.It is important to note that, although our results indicate promising antiviral potential, experimental validation is necessary to confirm the activity of the tested compounds of M. oleifera against SARS-CoV-2.The computational predictions provide a valid reason for in vitro and in vivo studies against SARS-CoV-2 of the herb M. oleifera.

Conclusions
Twenty-two compounds of M. oleifera showed inhibitory potential against SARS-CoV-2 proteins, which are crucial for virus infection and replication in host cells.Among them, ellagic acid, rutin, myricetin, quercetin, and luteolin were the most promising candidates that showed significant affinity with the S protein of the virus.Specifically, ellagic acid stood out as a promising candidate, demonstrating the best molecular affinity with the spike protein.This compound also demonstrated a better molecular interaction than the standard antiviral drugs approved by ANVISA for SARS-CoV-2.Furthermore, pharmacokinetic evaluations indicate that ellagic acid has satisfactory solubility and low toxicity, which ensures its viability as a therapeutic option for SARS-CoV-2 infection.It is also important to note that ellagic acid showed no evidence of skin sensitization or carcinogenicity in our in silico study.However, it is crucial to carry out experimental validations to consider these compounds as promising candidates for the treatment of COVID-19.

Figure 1 .
Figure 1.Molecular coupling of the ellagic acid ligand (red) with the spike protein results in a binding free energy of −9.3 kcal/mol (a) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black); (b) site of interaction of the protein-ligand complex; and (c) 3D conformation of the binding site of ellagic acid with the spike (S) protein.

Figure 1 .
Figure 1.Molecular coupling of the ellagic acid ligand (red) with the spike protein results in a binding free energy of −9.3 kcal/mol (a) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black); (b) site of interaction of the protein-ligand complex; and (c) 3D conformation of the binding site of ellagic acid with the spike (S) protein.

Figure 2 .
Figure 2. Total number of results presented in terms of binding energy (kcal/mol), organized by categories.

Figure 3 .
Figure 3. SARS-CoV-2 proteins that demonstrate high levels of interaction with Moringa oleifera compounds.

Figure 2 .
Figure 2. Total number of results presented in terms of binding energy (kcal/mol), organized by categories.

Figure 2 .
Figure 2. Total number of results presented in terms of binding energy (kcal/mol), organized by categories.

Figure 3 .
Figure 3.SARS-CoV-2 proteins that demonstrate high levels of interaction with Moringa oleifera compounds.

Figure 5 .
Figure 5. Molecular coupling of the ligand rutin (orange) with the ACE2 protein results in a binding free energy of −8.2 kcal/mol ((a) site of interaction of the protein-ligand complex; (b) 3D conformation of the binding site of rutin with the ACE2 protein; and (c) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black)).

Figure 5 .
Figure 5. Molecular coupling of the ligand rutin (orange) with the ACE2 protein results in a binding free energy of −8.2 kcal/mol ((a) site of interaction of the protein-ligand complex; (b) 3D conformation of the binding site of rutin with the ACE2 protein; and (c) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black)).

Figure 6 .
Figure 6.Molecular coupling of the isoquercitrin ligand (yellow) with the M pro results in a binding free energy of −8.9 kcal/mol ((a) site of interaction of the protein-ligand complex; (b) 3D conformation of the binding site of isoquercetrin with the M pro protein; and (c) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black)).

Figure 6 . 14 Figure 7 .
Figure 6.Molecular coupling of the isoquercitrin ligand (yellow) with the M pro results in a binding free energy of −8.9 kcal/mol ((a) site of interaction of the protein-ligand complex; (b) 3D conformation of the binding site of isoquercetrin with the M pro protein; and (c) 2D scheme showing hydrogen bonds (green) and hydrophobic interactions (black)).Adv.Respir.Med.2023, 91, FOR PEER REVIEW 14

Figure 7 .
Figure 7. Molecular coupling of the lutein ligand (green) with the RBD results in a binding free energy of −8.7 kcal/mol ((a) site of interaction of the protein-ligand complex; (b) 3D conformation of the lutein binding site with the spike/ACE2 complex and (c) 2D showing hydrogen bonds (green) and hydrophobic interactions (black)).

Table 1 .
Coordinates of the active sites of molecular targets.

Table 2 .
Results of the 244 dockings are carried out with the interaction of 61 ligands with ACE2, M pro , spike, and Receptor 5 of SARS-CoV-2. a Binding energy of the best conformation.

Table 3 .
Molecular affinity parameters of the chemical constituents of Moringa oleifera with ACE2, spike, M pro , and RBD of SARS-CoV-2.

Table 5 .
Absorption and distribution properties of Moringa oleifera compounds with the best molecular interaction energies.

Table 6 .
Metabolism and excretion properties of Moringa oleifera compounds with better molecular interaction energies.